The analysis of protein-containing solutions, in particular biological samples, often requires desalting of the solution for the removal of components that interfere with subsequent analytical techniques. Analytical techniques which may require desalting of sample solutions include those using component separation technologies such as electrophoresis, high performance liquid chromatography (HPLC) and mass spectrometry. Analysis of biological samples containing proteins using capillary electrophoresis, for instance, can be affected by the presence of various low molecular weight salts and protein degradation products. The presence of salts and other low molecular weight ions can increase the ionic strength of a sample to such an extent that the resolution upon electrophoresis decreases dramatically and/or the interpretation of the electropherograms becomes complicated due to the appearance of false peaks.
In addition to desalting, concentration of the sample is also often required since the particular component of interest may be present in small quantities. Frequently, the presence of a particular component in the sample will go undetected unless the component is concentrated to a level that is capable of being detected by the subsequent analytical technique. In the case of capillary electrophoresis, the concentration of the component in the sample may need to be substantially increased for the peak corresponding to the component on the electropherogram to be detectable.
The presence of proteins in samples of biological fluid, such as blood, serum, plasma, cerebrospinal fluid, tear, sweat, saliva and urine is a useful indicator of the presence or absence of certain disease states. Thus, the ability to identify and quantitate a variety of proteins in biological fluid can provide diagnosticians with information which may lead to the diagnosis of a variety of diseases. For example, determination of the urinary excretion of albumin, IgG, free protein human complex-forming glycoprotein (HC), alpha-1-macroglobulin, and kappa and lambda light chains allows in most cases for the classification of proteinuria in the clinically important categories. Using information obtained from the analysis of urine samples, for example, proteinuria may be classified as selective glomerular, unselective glomerular, tubular glomerular, or nontubular glomerular. The presence or absence of Bence Jones proteinuria may also be demonstrated.
The identification of monoclonal free light chains, or Bence Jones protein (BJP), is also important in the assessment of patients with multiple myeloma. Multiple myeloma may be characterized by the presence of BJP in the urine or serum. Approximately 13% of myeloma patients, however, have no BJP in their serum. Therefore, it is essential that both serum and urine be examined for the presence of BJP. The type of light chain present in the urine or serum sample may have a considerable effect on the clinical course of myeloma. For instance, lambda-type light chains are known to accompany rather aggressive myeloma, and exert their toxic effects in a shorter time than do kappa-type light chains.
Generally, identification of free light chains in urine is accomplished by analysis of urine samples with immunoelectrophoresis (IEF) or immunofixation electrophoresis (IFE). Both of these methods are labor intensive and require long execution times. Improved methods for identifying free light chains in urine involve capillary electrophoresis of samples in which the proteins of interest are subtracted from solution by being first fixed to a solid phase through antigen-antibody binding prior to electrophoresis. The identification of the specific proteins is established by their absence from the separated sample. This method is known as immunofixation electrophoresis by immunosubtraction, or IFE/s.
It is often preferable to use an instrument to analyze protein-containing solutions. The instrument should be capable of performing both routine serum protein electrophoresis (SPE) and follow-on, IFE/s testing to characterize monoclonal components detected in the initial SPE screening. One such instrument is the Paragon CZE.TM. 2000, available from Beckman Instruments, Inc., Fullerton, Calif. The Paragon CZE is a clinical, multi-channel automated capillary electrophoresis instrument. The unit is a dedicated analyzer for SPE and IFE testing, so that many of the traditional capillary electrophoresis parameters such as injection time, capillary rinse sequence and wash times, applied voltage, and absorbance wavelength selection have been optimized and automated to provide walk-away capability. Additional automated and optimized parameters of this instrument include sample volume in primary tubes, sample dilution and additional dilution with solid support. Using the Paragon CZE.TM. 2000 analyzer, the technique of immunosubtraction is used to remove a specific immunoglobulin class or type from a serum sample.
Regardless of the method used to identify proteins in solutions, and particularly urine, pretreatment of the solution to desalt and concentrate proteins in the sample solution is required. There are several classical methods for desalting or concentrating protein-containing solutions. These methods include dialysis, molecular sieve chromatography, diafiltration, ultrafiltration, precipitation, ion exchange, freeze drying, partitioning between two aqueous polymer phases, osmotic removal of water and reverse phase HPLC. These methods typically consist of two separate steps for desalting and concentration and may require specialized equipment and procedures associated with each step of desalting and concentration.
Gel filtration is one useful and mild method for desalting a protein-containing solution. The gel used in gel filtration processes consists of an open, cross-linked, three-dimensional molecular network, which can be cast in bead form for column packing and optimum flow characteristics. Pores within the beads are of such sizes that they are not accessible by large molecules, but smaller molecules can easily penetrate all the pores. Gel filtration media beneficially exhibit little protein binding and give high recoveries for even small amounts of proteins. Some of the most commonly used gel filtration support are Sephadex G-25 and BioGel P-30. Although gel filtration has many advantages, this method has several features which make it inapplicable for use in, for example, an ultrafiltration device. The primary problem with gel filtration media, particularly with polyacrylamide beads, is their softness. Even very gentle pressure, including osmotic pressures obtained during chromatography, can cause distortion, irregular packing and poor flow characteristics. Recently, Bio-Rad introduced the Bio-Spin chromatography column which is prepacked with a polyacrylamide gel matrix (Bio-Gel P-6). This ready-to-use column can be used for rapid desalting of a protein mixture at low centrifugal forces. Desalting in this manner, however, is only applicable for very small sample volumes and column volumes. Proper matrix preparation and column packing are required. The degree of desalting is further dependent on the column dimensions, shape and total volume. The optimal removal of salt that can be expected is achieved with sample volumes not exceeding 20-25% of the column volume.
Desalting of simple protein-containing solutions is often accomplished with ion exchange using, for example, a mixed bed resin. Commercially available mixed bed resins, such as IonClear BigBead (Sterogene Bioseparations, Inc., Arcadia, Calif.), AG 501-X8 (Bio-Rad, Hercules, Calif.) and Duolite mixed bed resin (BDH Laboratories, Poole, England; distributed by Gallard-Schlesinger Industries, Inc., Carle Place, N.Y.), are suitable for capturing both cations and anions from protein-containing solutions. Large bead size and surface modifications provide high capacity binding of small ions but very low capacity for much larger protein molecules. A mixed bed resin can be used in a packed bed column or mixed with the process feed to adsorb ions in the batch mode. In the case of complex biological samples, however, the pH of the sample treated with mixed bed resin can turn acidic and protein precipitation may occur. Accordingly, mixed bed resins are not suitable for the desalting of urine samples.
Another method for removal of salts from protein-containing solutions using ion exchange is accomplished using ion retardation resins. Methods using ion retardation resins, such as AG 11 A8 Resin (Bio-Rad, Hercules, Calif.) employ a different mechanism than conventional ion exchange in which salt uptake occurs via an actual exchange of ions. In the method using ion retardation resin, salts are adsorbed without an exchange of ions. The AG 11 A8 ion retardation resin has the ability to strongly bind hydrogen ions.
Jelkmann and Bauer used ion exchange chromatography with ion retardation AG 11 A8 Resin and mixed bed resin (AG 501-X8, Bio-Rad) to desalt and remove 2,3-diphosphoglycerate (DPG) from human hemoglobin. Jelkmann, W., Bauer, C., What is the Best Method to Remove 2,3-Diphosphoglycerate from Hemoglobin?, Analytical Biochemistry, 75, 382-388, 1976. They consequently used two columns with ion retardation and mixed bed resin to achieve this goal. The procedures involved multiple washing and eluting steps which are very time consuming.
Diafiltration is another method for desalting and concentrating protein-containing solutions. However, a one-step diafiltration process does not provide complete desalting. Only with multiple steps of diafiltration and changing buffers can one achieve the complete desalting necessary to analyze protein-containing solutions such as urine samples using capillary electrophoresis.
Hjerten et al. disclose a procedure for desalting and concentrating small volumes of biological samples, including urine. Hjerten et al., A Simple Method for Desalting and Concentration of Microliter Volumes of Protein Solutions with Special Reference to Capillary Electrophoresis, J. Cap. Elec. 1, 83-89, 1994. The method involves contacting microliter volumes of a sample with a polyacrylamide gel having a specified pore size and includes steps for preparation of the gel. The method yields a twenty-fold increase in protein concentration of the sample solutions. The combination of small sample yield and relatively low concentration increase after desalting and concentration makes this procedure impractical for the detection of proteins in protein-containing solutions which require 50-100 fold increase in concentration. The method is also inapplicable to automated systems for the analysis of proteins, particularly capillary electrophoresis instruments.
There is, accordingly, a need for a simple and efficient method for desalting and concentrating protein-containing solutions. A method is needed to remove irrelevant interfering compounds and concentrate proteins in the sample solutions for further analysis, for example, by capillary electrophoresis. In particular, a simple method for the desalting and concentration of urine proteins in a sample which is compatible with the subsequent analysis of the urinary proteins in a sample using an automated electrophoresis instrument is needed.